Transmembrane transport of ions and nutrients in yeast (Saccharomyces cerevisiae)

 

Our lab is interested in structure, function and regulation of molecular machines that mediate transport of small molecules across biological membranes. By combining the power of yeast genetics and the sensitivity of biophysical analysis, we are studying function of endogenous yeast membrane proteins (ion channels, cotransporter, ion pumps and water channels) in plasma membrane and tonoplast. For that we developed (adapted to yeast) and successfully applied methods – mainly patch-clamp – for recording electrical charge translocation on the level of individual transport proteins (ion channels), as well as on the macroscopic level, i.e. mediated by a whole population of transporters.

 

 

 

 

 

 

Endocytosis and exocytosis in yeast

 

Cells maintain physicochemical characteristics of membranes in order to allow for proper function of membrane-associated cellular processes, such as endocytosis and exocytosis. To investigate the interplay between membrane properties and biological processes, we applied lipid engineering approaches that allowed for systematic manipulation of fatty acid unsaturation and sterol biosynthesis, the main regulators of membrane fluidity. In combination with electrophysiological membrane capacitance measurements, we were able to study the dependence of the endo- and exocytic activity of Saccharomyces cerevisiae on membrane lipid composition in vivo. Our data suggest that lipid composition is intimately tied to membrane trafficking in yeast cells and suggest that endocytosis is particularly dependent on the lipid-defined properties of cell membrane.

 

 

 

 

 

Lipid composition and membrane function

 

The lipid composition of cell membranes and the interaction of lipids with the embedded membrane proteins can significantly influence membrane function and thus cell physiology. Formation of distinct membrane domains, trafficking and turnover rate of membrane proteins seem to be determined by the content of specific lipids. Physiological function and localisation of plasma membrane proteins are studied in engineered yeast strains in which the lipid composition of the membranes can be altered precisely by using inducible/repressible promoters controlling key genes in lipid biosynthesis. We are also exploring the existence/formation of microcompartments in the vacuolar membrane (see grey scale image)

 

 

CRISPR/Cas9 mediated genom modification

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The CRISPR/Cas9 technology has greatly improved genome editing in Saccharomyces cerevisiae over recent years. We constructed a new set of all-in-one CRISPR/Cas9 vectors that combine unique benefits of different already existent systems in order to further expand the technology’s design possibilities. Our vectors mediate constitutive gRNA expression whereas Cas9 expression is either driven from a constitutive or an inducible promoter. The introduction of desired gRNA targeting sequences into our inducible single gRNA vector relies just on in vivo homologous recombina- tion-mediated assembly of overlapping single-stranded oligonucleotides, thus reducing efforts of plasmid cloning to an absolute minimum. By employing the inducible system, yeast cells can be easily preloaded with plasmids encoding for a functional CRISPR/Cas9 system, thereby chronologically separating the cloning procedure from the genome editing step. Gene knockouts could be achieved with high ef ciency and effectivity by simply transforming preloaded cells with a selectable disruption cassette without the need of co-introducing any CRISPR/Cas9 system component. We also show the feasibility of efficient gene knockouts even when multiple gene copies were present such as in non-haploid strain backgrounds as well as the simultaneous deletion of two different genes in a haploid genetic background by using a multiplex variant of our inducible vector. The versatile applicability of our inducible vector system was further demonstrated by CRISPR/Cas9-mediated mating type switching of yeast.

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Yeast killer toxins

 

K1 is a yeast killer toxin of 19kDa, consisting in its mature form of two distinct subunits (alpha and beta), which are connected via three S-S bonds. The killer toxin is derived from a 35kDa preprotoxin, which is processed in the secretory pathway and exported from the cell via exocytosis. Although, K1 is the best studied yeast killer toxin, there is still an ongoing debate on the actual killing mechanism as well as on the cause of immunity of killer strains against their own toxin. Electrophysiological recording techniques, in particular the patch-clamp techniques are used to shed some light on these open questions.